I have a soluble receptor and I am trying to develop the binding assay conditions using radialebeled ligand. My problem is separating the bound from free radioligand. I've tried the precipitation method and it sort of works but I think I am loosing a lot of signal due to ligand dissociation during the precipitation time. I've also tried using dextran-coated charcoal but that is extremely not reproducible, in addition the charcoal doesn't completely remove the free radialigand and that severly effects the results.
Doe anyone know a better and quick method of separating free from bound radialigand for soluble receptors? Any references or advise would be appreciated,
Marek

galkam:

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Riad-Fahmy D, Read GF, Gaskell SJ, Dyas J, Hindawi R. A simple, direct radioimmunoassay for plasma cortisol, featuring a 125I radioligand and a solid-phase separation technique. Clin Chem. 1979 May;25(5):665-8.

Abstract:
A simple, direct radioimmunoassay for cortisol in human serum and plasma is described. An antiserum, raised in sheep to a cortisol-3-(O-carboxymethyl)oxime/bovine serum albumin conjugate, is coupled to microcellulose. No extraction is required because plasma samples and standards are incubated with the antiserum and an 125I radioligand in a low-pH buffer, which denatures cortisol-binding globulins. The assay satisfies accepted validation criteria. In addition, results from the radioimmunoassay compare well with those obtained by a gas chromatographic-mass spectrometric technique (r = 0.968; FRIA = 0.97 FGCMS + 2.0 nmol/L). The latter procedure features the very high intrinsic specificity obtained by selected ion monitoring at high mass-spectrometric resolution (M/deltaM = 8500) with a Varian MAT-731 instrument. The simplicity of the radioimmunoassay procedure, with use of reagents prepared "in house," makes this a very practical and economical assay for use in the medium or large endocrine laboratory.


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V. Breu, B. Butscha, and M. Clozel A Simple High Throughput FlashPlate Assayto Quantify ET-Antagonists in Plasma. F. Hoffman-La Roche Ltd., Switzerland.

Abstract:
Described is an assay combining liquid extraction and radioligand competition binding suitable for the quantification of endothelin (ET) antagonists in plasma, urine and tissue. The results of this assay, as obtained using conventional methods, are compared to those obtained using the FlashPlate platform
from PerkinElmer Life Sciences.

The assay system allows quantitative analysis of bosentan (a recently described potent, orally active ET antagonist on ETA and ETB receptors) in plasma of different species, including man, rat, rabbit, dog and marmoset. A lower detection limit of 20 ng/ml was reached using as little as 50 μl plasma, allowing pharmacokinetic studies in rats. The assay principle does not differentiate between parent compound and circulating active metabolites. Thus, by comparison with HPLC data, it is possible to estimate the contribution of active metabolites to activity. No circulating active metabolites were detected for bosentan using this methodology.

The assay was further developed on the FlashPlate platform, eliminating the extraction procedure, and resulting in an overall assay time of less than four hours, compared to approximately 24 hours using conventional extraction methodology. Using this improved method, the ET-receptor antagonists bosentan and SB 209670 could be quantified in rat plasma with detection limits of 15 and 4.5 ng/ml, respectively.


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Selleri S, Bruni F, Costagli C, Costanzo A, Guerrini G, Ciciani G, Costa B, Martini C. 2-Arylpyrazolo[1,5-a]pyrimidin-3-yl acetamides. New potent and selective peripheral benzodiazepine receptor ligands. Bioorg Med Chem. 2001 Oct;9(10):2661-71.

A new class of N,N-diethyl-(2-arylpyrazolo[1,5-a]pyrimidin-3-yl)acetamides (3f-y), as azaisosters of Alpidem, was prepared following a novel synthetic method and their affinities for both the peripheral (PBR) and the central (CBR) benzodiazepine receptors were evaluated. Binding assays were carried out using both [3H]PK 11195 and [3H]Ro 5-4864 as radioligands for PBR, whereas [3H]Ro 15-1788 was used for CBR, in rat kidney and rat cortex, respectively. The tested compounds exhibited a broad range of binding affinities from as low as 0.76 nM to inactivity and most of them proved to be high selective ligands for PBR. The preliminary SAR studies suggested some of the structural features required for high affinity and selectivity; particularly the substituents on the pyrimidine moiety seemed to play an important role in PBR versus CBR selectivity. A subset of the highest affinity compounds was also tested for their ability to stimulate steroid biosynthesis in C6 glioma rat cells and some of these were found to increase pregnenolone formation with potency similar to Ro 5-4864 and PK 11195.


NIH Molecular Imaging and Contrast Image Database - N,N-Diethyl-2-2(2-(4-[11C]methoxyphenyl)-5,7-dimethyl-pyrazolo(1,5-a)pyrimidin-3-yl)-acetamide[11C]DPA-713


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Hasler MJ, Painter K, Niswender GD. An 125I-labeled cortisol radioimmunoassay in which serum binding protein are enzymatically denatured. Clin Chem. 1976 Nov;22(11):1850-4.

Abstract:
We report an iodine-125 radioimmunoassay for cortisol in biological fluids, in which interfering binding proteins are enzymatically denatured. An antiserum to cortisol-3-carboxymethyloxime-bovine serum albumin, extremely low cross-reacting with other corticosteroids, was raised in rabbits. A cortisol-3-carboxymethyloxime tyrosine methyl ester derivative was synthesized and labeled with iodine-125 by standard radioiodination techniques. To eliminate the need for extraction and recovery procedures, we digested interfering binding witha proteolytic enzyme, which then was heat-inactivated before adding the labeled derivative and the premixed, preincubated antiserum complex. There was quantitative analytical recovery of esogenous cortisol added to sera from a normal man, a normal woman, and a pregnant woman. Values for the same samples agreed after extraction and chromatographic purification and agreed well with values obtained by other techniques by independent reference laboratories. The five-step assay can be done in 6 h or less.


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Kane JW. Use of sodium salicylate as a blocking agent for cortisol-binding-globulin in a radioimmunoassay for cortisol on unextracted plasma. Ann Clin Biochem. 1979 Jul;16(4):209-12.

Abstract:
This report describes investigations into the use of sodium salicylate as a cortisol-binding-globulin blocking agent and the subsequent development of a radioimmunoassay for cortisol on unextracted plasma. Cortisol antiserum was raised against a cortisol 3-O-(carboxy-methyl) oxime-bovine serum albumin conjugate. A 125I-labelled cortisol-tyrosine methyl ester conjugate was also prepared for use in the assay. The radioimmunoassay developed involved no pre-treatment of extraction of the samples before analysis and was extremely simple to perform. Comparison with another radioimmunoassay for cortisol and with the Mattingly fluorimetric assay gave good correlation.


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Brock P, Eldred EW, Woiszwillo JE, Doran M, Schoemaker HJ. Direct solid-phase 125I radioimmunoassay of serum cortisol. Clin Chem. 1978 Sep;24(9):1595-8.

Abstract:
We report a solid-phase iodine- 125 radioimmunoassay for serum cortisol in which the interfering binding proteins are inactivated by a combination of reaction pH and 8-anilino-1-naphthalenesulfonic acid. The procedure is easy to perform and gives accurate and reliable results. The shift in pH for optimum binding of an antibody resulting from immobilization on a solid support, used to decrease cortisol binding competition in the present test, is potentially exploitable in other antibody and enzyme systems.


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Dyas J, Read GF, Riad-Fahmy D. A simple robust assay for testosterone in male plasma using an 125I-radioligand and a solid-phase separation technique. Ann Clin Biochem. 1979 Nov;16(6):325-31.

Abstract:
A radioimmunoassay for testosterone in male plasma utilising a gamma-emitting radioligand and a solid-phase antiserum is described. The radioligand is testosterone-3-(O-carboxymethyl)-oxime coupled to 125I-iodohistamine, and the solid-phase antiserum is prepared by coupling antitestosterone-3-bovine serum albumin to cyanogen bromide activated cellulose. The new procedure retains much of the specificity associated with a published, specific radioimmunoassay using an antiserum raised against testosterone-11 alpha-BSA and a tritium radioligand and incorporating a dextra-coated charcoal separation procedure; values obtained by the two procedures are in excellent agreement (r = 0.98, n = 20). The combination of an 125I-radioligand and a solid-phase separation technique greatly increases sample throughput and has the further advantage of reduced running costs and a greater potential for automation. The method gives satisfactory levels of sensitivity, precision, and accuracy.



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Coldwell RD, Nolan J, Trafford DJ, Mawer EB, Makin HL. The use of octadecyl-bonded microparticulate silica in the separation of free and bound fractions during saturation analysis of vitamin D metabolites. J Pharm Biomed Anal. 1992 Sep;10(9):631-7.

Abstract:
The use of octadecyl-bonded microparticulate silica to separate free and bound fractions during the saturation analysis of 25-hydroxyvitamin D and 1,25-dihydroxyvitamin D has been investigated. A slurry of octadecyl-bonded silica in an appropriate incubation buffer was prepared and used in parallel with a conventional dextran-coated charcoal suspension in several assay procedures. Standard curves, non-specific binding and plasma values were compared. A competitive protein binding assay for 25-hydroxyvitamin D and two radioreceptor assays and one radioimmunoassay for 1,25-dihydroxyvitamin D were investigated. In most cases the octadecyl-bonded silica preparation gave the more favourable results; its action was rapid, time- and temperature-independent, and it produced low non-specific binding and higher B0 values in all the assays examined. It was in our hands easier to use than dextran-coated charcoal. The use of octadecyl-bonded silica is recommended as an efficient agent for the separation of free and bound fractions in the saturation analysis of vitamin D metabolites.



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Screen Design and Assay Technology Special Interest Group
Monday, September 13, 2004
Chair: Usha Warrior and co-chair, James Kofron
Outline: Main Targets and Assay Technology
Advantages and Disadvantages of assay formats

Society for Biomolecular Sciences Special Interest Groups - Screen Design & Assay Technology Special Interest Group



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